44
Virology practice Dr. Y. Hamadt Allah Elhaj Ass. Prof. medical microbiol ogy

Virology practice

Embed Size (px)

DESCRIPTION

Virology practice. Cultivation of viruses. Since viruses are obligate intracellular parasites they have to be grown in living cells. There are three systems for their cultivation: 1- tissue culture. 2- embryonated eggs. 3- intact animals. 1- tissue cultures: - PowerPoint PPT Presentation

Citation preview

Page 1: Virology practice

Virology practice Dr. Y. Hamadt Allah Elhaj

Ass. Prof. medical microbiology

Page 2: Virology practice

Cultivation of viruses • Since viruses are obligate intracellular

parasites they have to be grown in living cells.• There are three systems for their cultivation:1- tissue culture.2- embryonated eggs.3- intact animals.1- tissue cultures: Pieces of animal or human tissues are

trypsinized to get separate cells. Thses are grown in presentceof growth medium containing serum, on glass or plastic tubes, bottles or plates with a flat side.

Page 3: Virology practice

A monolayer or sheet of cells is formed on the

flat side of container within few days. Viruses

are inoculated on the monolayer. There are

three types of tissue cultures:

1. Primary cell lines: these are prepared from

organ fragments e.g. monkey kidney.

such cells can only divide for several

passages (4 - 6) and then degenerate.

2. Continuous cell lines: these are derived

from tumour cells and they can divide

indefinitely e.g. hela cells which are derived

from carcinoma of the cervix.

Page 4: Virology practice

3. Human diploid cell lines: these

are usually fibroblasts derived

from human embryo tissues .

They have diploid number of

chromosomes. They grow rapidly

and can be subcultured up to

about 50 passages in culture e.g.

human embryo lung tissue.

Page 5: Virology practice

Detection of virus replication in cell

culture:

1- cytopathgenic effects (CPE): these are

changes in cells that can be observed

microscopically:

a. Cell death and detachment from the glass

surface is produced by many viruses e. g.

poliovirus.

b. Rounding and grape like cluster formation

is produced by adenovrus.

c. Cyncytium or multinucleated giant cell

formation are characteristic of measles or

mumps.

Page 6: Virology practice

d. Cell transformation: the cells lose

the property of contact inhibition

present in normal cells and pile

up to form foci of malignantly

transformed cells e.g. tumour

viruses.

Page 7: Virology practice

2- Plaque formation: plaques are virally

infected areas in tissue culture monolayer.

They can be seen by the naked eye as

unstained areas when using vital stains.

3- Inclusion bodies: these are intranuclear or

intracytoplasmic structures which may

appear in virus infected cells and can be

seen by the light microscope. They are

often the site of virus replication. Their

presence is of diagnostic value e.g. the

(Negri bodies) in nerve cells of rabid

animals.

Page 8: Virology practice

4 -Haemadsoption:When RBCs are added to infected cells they will appear as rosettes or clumps on the areas where the virus is growing. This is useful in haemagglutinating viruses such as influenza virus.5- Fluorescent antibody staining: infected cell sheets on cover slips or microtiter plates may be treated with fluorescein labelled specific antibody and examined for positive fluorescence.

Page 9: Virology practice

6- Interference: in some viruses which

do not produce CPE their growth can be

proved by their ability to interfere with

the growth of another CPE producing

virus.

7- detection of viral antigens by

serology: including complement

fixation test, haemagglutination test ,

…..

Page 10: Virology practice

8- neutralization tests: neutralization of

effects of virus on tissue culture by

specific antisera can be used to identify

and type the virus isolated.

Page 11: Virology practice
Page 12: Virology practice
Page 13: Virology practice
Page 14: Virology practice

Plaque

Page 15: Virology practice
Page 16: Virology practice

CPE: inclusion bodies

Negribodies

Negribodies

neuron

Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia

Fig. 39.9)

Page 17: Virology practice

Hemadsorbtion

add red blood cells

Page 18: Virology practice

BEST WISHESDr. yagoub Hamadt

Allah

Page 19: Virology practice

2- embryonated eggs:Different viruses can grow in various cavities of embryonated eggs or in the developing embryo itself. The age of the embryo used and the site of inoculation vary according to the virus inoculated.Chorioallantoic membrane inoculation is used in pox and herpes viruses.The influenza virus can readily grow in the amniotic sac and in the respiratory cells of the embryo.

Page 20: Virology practice

3- intact animal:Animal inoculation was mainly used in the past when tissue culture methods were not known, however, animal inoculation is still used for studying viral oncogenesis, pathogenesis of viral disese, immune response to viruses and for primary isolation of some viruses.The white sucking mouse is the most widely used; it is susceptible to encephalitis viruses by the intracerebral route.

Page 21: Virology practice

Diagnosis of virus infectionthe laboratory procedure used in diagnosis of viral disease include:a. Direct detection of viruses or their antigens in clinical specimens.b. Isolation of viruses.c. Serologic detection of antiviral antibodies.

Page 22: Virology practice

a. Direct detection of viruses or their antigens in the

specimens:

Can be achieved by different techniques:

1- Light microscope: this can be used to visualize some

large viruses e.g. poxviruses in which elementary bodies

can be seen in skin lesions (papules and vesicles).

Inclusion bodies can also seen under the light microscope

in several viral infections. In rabies, intracytoplasmic

inclusions called (negri bodies) can be detected in nerve

cells.

Page 23: Virology practice

2- Electron microscope: is used to demonstrate virus

particles in vesicular fluid or tissue extracts treated with

special stains. It is only successful if large numbers of

particles (109/ml) are present.

3- Immunoelectron microscope (IEM): addition of

specific antisera to the clinical material leads to

aggregation of virus so it can be detected more readily

than separate virus particles e.g. diagnosis of HAV and

rota virus in stool.

Page 24: Virology practice

4- Immunofluorescence microscopy: detection of virus in

smears from lesions using fluorescein labeled specific

antisera. E.g diagnosis of rabies virus in brain smears.

5- Solide – phase immunoassay: both radioimmunoassay

(RIA) and enzyme linked immunosorbent assay (ELISA)

can be used for detection of viral antigens in different

clinical specimens such as detect of HCV, HBV,

rotaviruses,….

Page 25: Virology practice

6- Nucleic acid hybridization: using DNA probes, it is possible to

detect virus nucleic acid in pathologic specimens or in tissue

samples. The probe which is a single strand of the nucleic acid of

the virus in question will hybridize with its complementary strand in

the specimen. Probes are labeled and can be easily detected.

7- Polymerase chain reaction (PCR): is a recent technique which

involves amplification of a short sequence of a target DNA or RNA

( which may be in low concentration e.g one copy). Leading to

accumulation of large amount of that sequence, so it can be easily

detected.

Page 26: Virology practice

b. Isolation of viruses:

Isolation of viruses from clinical

specimens by inoculation on

tissue culture, chick embryo or

laboratory animals according to

the virus in question.

Page 27: Virology practice

c. Serologic detection of antiviral antibodies:

Serologic diagnosis of virus infections can be

established by detecting a rising antibody titre to

the virus. the first sample should be collected

early after onset, the second 1 -2 weeks later.If paired sera are not available or rapid diagnosis is needed, as in diagnosis of rubella in early pregnancy; detection of IgM antibodies to the virus is resorted to. The detection of IgM in a single serum sample, indicates recent infection.

Page 28: Virology practice

The tests used for serologic diagnosis of

viral infection include:

1- Virus neutralization

2- Complement fixation

3- Haemagglutination inhibition.

4- Immunofluorescence.

5- RIA 6- ELISA

Page 29: Virology practice

Neutralization test

This is based on

the neutralization of the

virus infectivity by mixing it

with specific antibody before

inoculation into

cultures.

Page 30: Virology practice

1 2 3

B

Following virus isolation:

1.Divide culture yield into small volume in a set of test tubes

2. Prepare the panel of antisera against which the virus isolate is to be challenged

2. To each test tube add one antisera and leave one as a virus control and one as serum control

Page 31: Virology practice

Incubate for one hour then inoculate each into cell culture tubes, incubate and observe daily.

Page 32: Virology practice

2 31

Principle of Neutralization test

Page 33: Virology practice

Viral Hemagglutination Inhibition Test

• Useful for viruses that aren’t cytopathic• Test based on viral hemagglutination,

the ability of some viral surface proteins to clump red blood cells

• Serum from an individual will stop viral hemagglutination if the serum contains antibodies against the specific virus

• Commonly used to detect antibodies against influenza, measles, and mumps

Page 34: Virology practice

Complement Fixation Test

• Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes

• Used to detect the presence of specific antibodies in an individual’s serum

• Can detect antibody amounts too small to be detected by agglutination

Page 35: Virology practice
Page 36: Virology practice

Fluorescent Antibody Test

• Uses fluorescent dyes as labels • Fluorescein is the most important dye used

in these test– Chemically linked to an antibody without

affecting antibody’s ability to bind antigen– Glows bright green when exposed to fluorescent

light

• Fluorescein-labeled antibodies used in two types of tests– Direct fluorescent antibody test– Indirect fluorescent antibody tests

Page 37: Virology practice

Direct Fluorescent Antibody Tests

• Identifies the presence of antigen in tissue– Tissue sample flooded with labeled antibody– Antibody and antigen are allowed to bind for a

short period– Unbound antibody washed from the preparation– Results observed under a fluorescent microscope

• Used to identify small numbers of bacteria in patient tissues

• Not a quantitative test – the amount of fluorescence observed is not directly related to the amount of antigen present

Page 38: Virology practice

Indirect Fluorescent Antibody Tests

• Can be used to detect antigens in cells or patient tissues

• Also used to detect specific antibodies in serum via a two-step process

Page 39: Virology practice

Indirect Fluorescent Antibody Test

Figure 17.14

Page 40: Virology practice

ELISA

• Stands for enzyme-linked immunosorbent assay

• Uses an enzyme as the label– Reaction of the enzyme with its

substrate produces a colored product indicative of a positive test

• Most common form of ELISA is used to detect the presence of antibodies in serum

Page 41: Virology practice

ELISA

Page 42: Virology practice

Antibody Sandwich ELISA

• Modification of the ELISA technique• Commonly used to detect antigen• Antigen being tested for is

“sandwiched” between two antibody molecules

Page 43: Virology practice

Antibody Sandwich ELISA

Page 44: Virology practice

Advantages of the ELISA

• Can detect either antibody or antigen• Can quantify amounts of antigen or

antibody• Easy to perform, inexpensive, and

can test many samples quickly• Plates coated with antigen and

gelatin can be stored for later testing